1. Field of the Invention
The invention is related to methods and apparatus to separate, and isolate for testing, serum or plasma, red cells, and white cells in evacuated blood collection tubes; obtaining oil free plasma or serum; and harvesting of white cells.
2. Brief Description of the Prior Art
Human blood is routinely collected in sealed, evacuated test tubes and centrifuged to separate the lighter serum or plasma portion from the heavier red blood cells. Typically a portion of the serum or plasma is then removed and tested. While the separated blood is stored awaiting testing certain chemicals can migrate between the separated layers giving incorrect test results. Consequently, the art has developed a variety of separators having a specific gravity between the serum or plasma and the red blood cells. These separators are either solid devices or gels.
The first solid separator was disclosed in U.S. Pat. No. 3,508,653 to Coleman. That device was a rubber or other elastomeric cylinder. A major problem with that device was the inability to maintain a seal because it is costly to maintain the precise inner diameter of the test tube when mass produced. The separator of Lawhead's U.S. Pat. No. 3,814,248 was the next solid separator development following Coleman's invention. Lawhead's separator is a centrifugally motivated spool originating from and placed adjacent to a vacuum maintaining stopper. There is also a polystyrene sphere free and unconstricted within the hollow inside of the tube. In use, blood is collected in the tube and is centrifuged. That action induces the free sphere to move under centrifugal force to join the socket-like underside of the floating rubber spool at the interface of the heavy and light phases. This product was unable to be used for the harvesting of serum because the floating ball frequently became enmeshed in the clot, preventing enclosure of the valve-like separator. Further, the separator did not seal firmly enough to withstand more than mild shock to the tube of separated blood, resulting in remixing of plasma and cells during transport, and other handling.
Ayres' U.S. Pat. No. 3,779,383 describes a complicated, costly device in which the blood introduction end of the tube is opposite to the movable separator end of the tube, and abutting an impenetrable rubber closure. Ayres' embodiment was unable to provide interface separation of the light and heavy phases of the blood because the separation element did not possess a specific gravity intermediate the two phases, but relied on an arbitrary stop element molded into the collection tube, combined with a centrifugally operated element having a specific gravity considerably higher than the blood cells. Blaivas was issued U.S. Pat. No. 3,786,985 for a device very similar to the Ayres' collection tube, excepting that the centrifugally motivated separator element abutted the needle penetrable stopper.
North in U.S. Pat. No. 3,931,018 discloses a solid separator for use in separation of blood serum and blood plasma using centrifugal force that must be inserted into the blood collection tube after blood collection. This device has a disc filter element above the solid separator having a specific gravity intermediate the light and heavy phases. The product has not attained significant acceptance because it is less convenient and less efficient to use and compromises sterility. Others have made centrifugally motivated solid separator devices of various configurations including a hollow, piston-like, coaxial tube disclosed in U.S. Pat. No. 4,159,896 to Levine et al., or ring disclosed in U.S. Pat. No. 5,236,604 to Fiehler, a closed end coaxial tube disclosed in U.S. Pat. No. 4,946,601 to Fiehler, an umbrella shaped solid separator device designated to ascend to the interface during centrifugation disclosed in U.S. Pat. No. 4,152,270 to Cornell, and a dual component assembly having a rigid conical core surrounded by a cup-shaped elastomeric component disclosed in U.S. Pat. No. 4,877,520 to Burns. Tubes containing these separators are limited in their function, and cannot be used for transport, or with many of the newer large high through-put analyzers.
J. A. McEwen, et al, in U.S. Pat. No. 5,030,341, disclose a solid separator system in which blood collection tubes are rotated on their axis to centrifuge the contents, rather than in the conventional way in which a blood filled tube is rotated with the tube's axis and contents rotate perpendicular to the centrifuge's axis of rotation. Such a system does not appear to be economical, since a complete change in equipment requirements, particularly the centrifuge device must be made. The system also requires relatively expensive disposable collection tubes.
As a result of the difficulty in designing what has been considered a commercially viable solid separator, manufacturers have resorted to the use of semi-solid, or thixotropic gel materials. A thixotropic gel is used in this context to mean a material formulated to remain rigid enough to remain in place, i.e., "firm", or "solid", until an external stress, such as a centrifugal force is applied. In a vacuum collection tube containing blood and gel, centrifugation of the tube at the proper level of acceleration (g force) would cause the gel to deform, flow to the interface between the packing cells and the lighter liquid phase, and finally come to a stable settled state reformed into a disc or cylindrical segmented barrier between the heavy phase, and the light phase.
Lukacs and Jacoby (U.S. Pat. Nos. 3,780,935 and 3,963,119) introduced the use of a very viscous silicone separator material prepared by intimate mixing of silicone oil and amorphous silica in proper proportions to yield a highly viscous sealant material having a specific gravity in the range between the specific gravity of the light phase and the specific gravity of the heavy phase of blood. They claim the silicone material in these patents, used with a funnel-like device inserted into the tube of collected blood, flows into the centrifugally separating blood, forming a barrier layer of viscous silicone oil and silica material with a specific gravity about 1.03 to 1.05. This product, sold under the trademark "SURE-SEP", provides a poor seal because the composition is not a firm gel and flows under gravitational force alone. It has the great disadvantage of needing to be inserted after the blood has been collected and not being an element assembled and integrated into the blood collection tube.
A further approach is the addition of granulated or powdered materials having a specific gravity intermediate the light and heavy blood phases to tubes of collected whole blood, followed by centrifugation, for separation of the heavy and light phases of the blood. Use of insoluble small particles of the desired specific gravity first described by Nishi in 1965, Clin. Chem. Acta, 11 (1965) pp. 290-292. Another modification of this technique was patented by Adler in U.S. Pat. No. 3,647,070. Adler added water-swellable, non-ionic hydrophilic polyhydroxyethylmethacrylates or polyacrylamide hydrogels in granulated or disc form, with a specific gravity between the two centrifuged, separated phases. These materials were added to blood collection tubes only after centrifugation to provide for a barrier between the heavy and light phases. Neither case provides for an integrated blood collection tube that permits a separation of whole blood collected, without the transference of plasma through the interstices. The blood corpuscles squeeze through the gaps between the solid particles, even when swollen and compacted after centrifugation. Further, the granulated separator mass is readily broken by impact, or unusual movement, and the container must be protected from shock during transport.
Zine, received U.S. Pat. No. 3,852,194 for the apparatus and method of using a silicone oil containing a silica thixotropic composition in a vacuum blood collection tube. The tube assembly with the silica/silicone oil gel composition in a vacuum collection tube uses an energizer plunger to move the gel toward it's sealing position between the separated phases. Subsequent to that patent there has been much development of an incremental nature intended to formulate many subspecies of silicone gels having desirable properties such as radiation stability. These efforts have generally been directed to mixing silicone oil with amorphous silica, and often a surfactant for gel network formation and maintenance. Usually, hydrophobic amorphous silica particles were used to provide optimal conditions of stability of the thixotropic material, along with the desired specific gravity, and stabile viscosity. Other manufacturers later replaced the original dimethylsiloxane (silicone) oils with other organic oils to compound thixotropic gels from amorphous silica.
Difficulties in providing silicone gel commercial products, have caused abandonment of this material for use in blood separator assemblies by Sherwood, Becton Dickinson, and the original group that introduced the Lukacs and Jacoby products, General Diagnostics. U.S. Pat. Nos. 4,021,340 and 4,180,465 have been issued for thixotropic blood separator gels using polybutene in lieu of dimethylsiloxane oil for use in mixing with amorphous silica. Becton Dickinson replaced silicone oil based gels around 1980 using gels based on polyester oil in the "SST" product following the teaching of Lamont and Braun in U.S. Pat. Nos. 4,101,422 and 4,148,764. This polyester gel, and virtually all commercial thixotropic gels, require mixing an oil with amorphous, usually hydrophobic silica to adjust specific gravity and to ensure thixotropy. Following the same trend of mixing a base oil with silica, the Terumo Corporation obtained U.S. Pat. No. 4,172,803 for a gel containing four additional butene related polymers.
Okuda, Abo, and Shinohara were issued U.S. Pat. No. 4,140,631 for a sealant comprising alkyl acrylates or methacrylates as sealants, claiming a significant advantage of clarity, and ability to dispense with the need for amorphous silica. In this case a high viscosity Newtonian sealant is the result. If amorphous silica is added the resultant sealant becomes a thixotropic gel which will be practically transparent.
Mendershausen has demonstrated the existence of serious problems associated with the use of gel compositions of oil and silica when used as separators in blood collection tubes. In the case of certain Eastman Kodak Products, oil globules have been seen in the light phase of the blood and oil films at the top of the light phase often seriously affect results of blood glucose ("blood sugar") tests. The oil can form a film on the test slides, causing blocked diffusion of the substance being analyzed into the slide.
In one Boehringer-Mannheim product, the BMC/Hitachi 747 Chemistry Analyzer, it was found that ion specific electrodes can cause reporting of spurious sodium levels in patients, because polyester oil droplets originating from gels float freely in the light phase of separated blood, or form oil films over the light phase of the blood. The oil is thereupon transferred onto electrode probes that are immersed into the light phases of the blood for testing. Mendershausen believes that the oil coating on the surface of the sodium electrode probe insulates the electrode, changing the electrical potential, and consequently the reported values of the sodium. Such changes on the sodium electrode can affect other electrodes (potassium, calcium, chloride, etc.) and can lead to grave problems resulting from the inaccuracies reported.
Separation of oil from the gel in blood separator tubes of oil has been noted by engineers in Sherwood assigned patents, earlier by Murty, and subsequently by Fiehler (U.S. Pat. Nos. 4,180,465, 4,946,601, 5,236,604, and 5,269,927) Solutions offered ranged from use of polybutene oil in place of silicone oil, isolation of the gel prior to blood collection, and the use of plastic oil capturing devices. In all the disclosures, the importance of the problem was restated and reoutlined, but no satisfactory solutions were provided because the root cause was not eliminated. Inevitably, blood separation tubes containing gel released oil during centrifugation, because all the gels comprise an inherently unstable mixture of two phases of materials, one is heavier than the heaviest phase of the blood, while the other (the oil) is lighter than the lightest phase of the blood and rises to the top.
Burns in U.S. Pat. No. 4,877,520 provides additional support for the need to replace gel separators with solid separators. At Column 1, lines 60-66, he states, "Moreover, the shelf-life of the product is limited in that globules are sometimes released from the gel mass or network. These globules have a specific gravity that is less than the separated serum and will float in the serum and can clog the measuring instruments, subsequently, during the clinical examination of the sample collected in the tube."
The use of silicone separator gel in evacuated blood collection tubes preceded a number of patents dealing with separation of lymphocytes and monocytes blood containing anticoagulants for testing purposes. A. A. Luderer et al, in U.S. Pat. Nos. 5,053,134 and 4,190,535, and W. C. Smith et al, in U.S. Pat. Nos. 4,957,638, 4,954,264, 4,867,887, and 4,844,818 all basically use gel separators of defined specific gravity to separate and isolate the mononuclear white cellular elements from the red cells of the blood. Many add other components to the gel base. These methods are feasible, but they are not necessarily simple, rapid, or particularly well suited to automation.
Wardlaw, et al, in U.S. Pat. Nos. 4,027,660 and 4,077,396 et seq., describe a blood testing device which expands the axial presentation of white blood cells in a small bore tube of blood upon centrifugation. This was used to provide a simple, rapid means to determine both total, and differential white blood counts. Such an invention has become the basis of widely used blood counting systems, often in a physician's office laboratory. Such a device is designed for diagnostic purposes only, since no means are provided to collect the various white blood cells separated in the system. Further, the devices contain stain to differentiate the type of white cells present in the tubes.
In our U.S. Pat. No 5,065,768 we disclose tubes with self-sealing plugs having an air vent channel which automatically seals a few seconds after the blood sample contacts the plug. The blood sample may then be dispensed with aid of a special pipette, or centrifuged in a microhematocrit or functionally similar centrifuge at about 11,000 g after collection of the fluid. This provides a packed cell volume reading, which may be followed by plasma dispensation from that tube. This invention is directed primarily toward the collection of blood from fingersticks, and discloses self-sealing plugs that seal off the air vent as a direct result of contact with the specimen at the time the sample is filled with blood from skin punctures. This invention does not relate to sealing devices that are intended to begin to seal during and after centrifugation has begun. Furthermore, the patent teaches that solid separators or gels are used to separate the phases of centrifuged blood.
Walder et al. in U.S. Pat. No. 5,322,659 disclose a two-layer tubing having a hydrophilic polymer outer layer. The tubing is dipped into a solution that carries anti-infective reagents or other agents which are absorbed by the polymer layer. There is no concern with a change in dimension after absorption of the solution. The tubing is then inserted into the body and there releases the reagent. These tubes are not intended or suitable for use as sealing devices.
There is a need for a simple, effective, and economical solid device for separation of serum and plasma. There is also a need for a solid separator to facilitate the harvesting of lymphocytes and monocytes in contemporary clinical laboratory testing for a multitude of immunological, genetic, microbiological, and other testing purposes as well as to aid collection of platelets and small white cells used in coagulation studies.